Currently, potential eye and skin irritation of many chemicals, household cleaning products, cosmetics, paints and other materials are evaluated through direct application to animals or human subjects. A common way of measuring the irritancy and effect of material on the eye or skin is through the Draize test in which a material is applied directly to a rabbit's eye or skin and the irritation measured. (Draize, J. H., Woodward, G. and Calvery, H. O. (1944), "Methods for the Study of Irritation and Toxicity of Substances Applied Topically to the Skin and Mucous Membranes", J. of Pharm. and Exp. Therapeutics, 82; 377-390). A low volume test for eye irritation has been devised but this still requires living subjects.
Previously a skin keratinocyte Neutral Red assay method for in vitro assessment of skin Irritation was developed for testing chemical ingredients. (Osborne, R. and Perkins, M. A. (1990), "In Vitro Skin Irritation Testing with Human Skin Cell Cultures", Toxic in Vitro 5, 563-567, U.S. patent application Ser. No. 07/647,379, filed Jan. 28, 1991). These keratinocyte cell cultures are submerged in an aqueous buffered medium during the testing. Therefore any material which is added to the culture must also be soluble in the buffered medium used to grow this culture. Any test materials must be compatible with water and able to be diluted. Therefore, these systems are limited in their ability to predict the irritancy potential for aqueous incompatible materials, solid or gel-like product formulations, and acids or bases which would react with the buffer. Moreover, cultures which are submerged in a buffered medium cannot mimic the in vivo topical application methods in which neat materials are applied directly to the eye or skin surface.
Therefore, there is a need to provide a method of evaluating topically applied neat materials in vitro. A method in which materials could be placed directly onto the cell culture and the effect or irritation of the material studied over time is highly desirable. It is an object of the invention herein to provide a method for topical application of neat (undiluted) test materials as an alternative model for eye and skin irritancy testing. Skin cultures without a stratum corneum that have a histologic similarity to both eye and skin morphology are used. The cytotoxicity of test materials to the cells is determined by MTT assay. MTT assay for cell viability is based on the reduction of a tetrazolium dye by functional mitochondria. Other biochemical endpoints such as the release of the cytosolic enzyme lactate dehydrogenase (LDH), and the inflammatory mediator protaglandin E.sub.2 (PGE.sub.2), can be measured. These markers correlate with human skin irritation responses.
It is an object of this invention to provide an alternative method to eye and skin irritation testing in rabbits or other living models that is able to predict human irritancy potential of all types of formulations, including solid, granular and gel-like household cleaning products and cosmetics.
It is another object of this invention to provide novel topical application methods for problem test materials for example, gels, foams, pastes, granular materials, creams, solids, powders and acids or bases. It is a further object to provide a method which works for aqueous incompatible test materials.